Recent evidence has suggested that inconsistencies in human islet yield and viability after collagenase digestion is attributed to the activation of endogenous enzymes of the cadaveric donor pancreas. A study of the enzyme kinetics of serine proteases throughout human islet isolations showed a significant increase in activity levels throughout the digestion period. Following the digestion, it is important to further inhibit these enzymes by the addition of an inhibitor to the dilution media.
To report the levels of endogenous pancreatic enzymes remaining after human islet isolation and the effects of three potential enzyme inhibitors on the proteases.
Human albumin, fetal calf serum, and the protease inhibitor aprotinin were incubated with the trypsin, chymotrypsin, elastase, and collagenase and were assayed for activity.
Results at the final stage indicated that chymotrypsin retained 21.0 ± 7.5% (mean ± SE; n = 20) of the activity observed at the conclusion of the enzymatic digestion phase of the isolation process, whereas trypsin, elastase, and collagenase retained 3.0 ± 1.5%, 2.1 ± 0.6%, and 3.9 ± 0.9%, respectively. Fetal calf serum and aprotinin showed strong inhibitory effects against bovine pancreatic trypsin; however, they showed a weak inhibitory effect against elastase. Supplementation with aprotinin failed to inhibit human chymotrypsin and elastase. Human albumin showed minimal inhibition and was shown to serve only as a competitive inhibitor. No inhibition to collagenase was observed with human albumin, fetal calf serum, or aprotinin.
This study clearly demonstrates that low amounts of endogenous pancreatic enzymes remain active throughout the human islet isolation process and that the added inhibitors at the end of the isolation process are not fully effective at inhibiting the enzymes.
*Department of Surgery, Surgical-Medical Research Institute, and †Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
Manuscript received November 21, 2002;
revised manuscript accepted February 6, 2003.
Address correspondence and reprint requests to Dr. Jonathan R. T. Lakey, Director Clinical Islet Laboratory, Assistant Professor of Surgery, Department of Surgery, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Center, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada. E-mail: firstname.lastname@example.org